7. Project Summary/Abstract Coxiella burnetii, the causative agent of Query (Q) fever in humans and coxiellosis in other species is a highly infectious obligate intracellular bacterium. In humans, this disease is largely self-limiting, presenting as an acute febrile disease; however, the pathogen is capable of causing both hepatitis and potentially life- threatening endocarditis. C. burnetii is capable of infecting a broad range of eukaryotic cell types within which the bacterium replicates exclusively in a host-derived compartment referred to as the Coxiella Containing Vacuole (CCV). CCV biogenesis is dependent on C. burnetii secretion of Type IV secretion system (T4SS) effector molecules and in their absence the bacterium is unable to replicate intracellularly and/or exhibit virulence. Studies on the physicochemical requirements of C. burnetii have established that the moderately acidic pH of the CCV, a phagolysosome-like vacuole, dictates C. burnetii nutrient transport, protein synthesis and replication. Additionally, C. burnetii exhibits tropism for tissues directly involved in iron storage and recycling (e.g., the liver and splenic red pulp). High iron loads in the liver and splenic red pulp, and known uptake of extracellular material into the CCV lumen by fluid phase endocytosis, is consistent with uptake of iron-containing molecules (e.g., transferrin) into the CCV. Cultured host cells infected with C. burnetii have been shown to increase expression of transferrin receptors, suggesting a demand for iron, a micronutrient previously reported to have a limited role in C. burnetii pathogenesis. While genome sequence analysis suggests C. burnetii has a limited capacity to acquire iron via siderophores or uptake systems for iron- containing molecules, the C. burnetii genome does encode the ferrous iron uptake transporter FeoAB suggesting that molecular iron is the natural iron source for C. burnetii. Additionally, the C. burnetii genome encodes the transcriptional regulator stringent starvation protein A (SspA) that in Escherichia coli has been shown to positively regulate genes involved in acid tolerance. Since replication is essential for virulence, and replication is dependent on both moderately acidic pH and iron, we hypothesize that pathogen replication is triggered by simultaneous sensing of niche-specific variables (i.e., acidic pH and iron) that co-regulate expression of virulence factors required for CCV biogenesis and pathogen growth.